Progesterone is a steroid essential for the maintenance of normal pregnancy in mammals, including primates. Among other things, progesterone: (a) causes development of decidual cells in the uterine endometrium which are important in nutrition of the early embryo; (b) increases secretion of nutritive substances in the uterus and fallopian tubes to aid in the development of the zygote prior to implantation; and (c) reduces the risk of spontaneous abortions via a decrease in contractility of the uterus. Thus, removal of progesterone from circulation before implantation or nidation (embedding of the early embryo into the uterine mucosa) can prevent implantation whereas removal after nidation can prevent normal maintenance of pregnancy.
Progesterone is mainly, although not exclusively, metabolized in the liver to the principal metabolite, 5.beta.-pregnane-3.alpha.,20.alpha.-diol. While 5.beta.-pregnane-3.alpha.,20.alpha.-diol is the principle metabolite, progesterone is also metabolized by the ovaries. Specifically, the 20-oxo group is reduced to, among other substances, 20.beta.-hydroxypregn-4-ene-3-one (i.e., 20.alpha.-hydroxyprogesterone). 20.alpha.-hydroxyprogesterone, unlike progesterone, is biologically inactive and cannot maintain pregnancy or even allow implantation to occur [Talwalker et al., Endocrinology 74:86-87 (1966); Wiest et al., Endocrinology 82:844-859 (1968)].
Presently, the only safe abortifacient available is the progesterone antagonist, mifepristone (RU 486). When administered early in pregnancy, mifepristone's main mechanism of action is to cause decidual breakdown by blockade of uterine progesterone receptors. However, for mifepristone to cause a complete abortion, a prostaglandin must be administered approximately 48 hours after mifepristone administration to further increase uterine contractions and ensure expulsion of the detached blastocyst. Therefore, there remains an interest for a novel substance that can not only induce abortion but also prevent implantation and pregnancy without the side effects or drawbacks of the currently available agent.
The enzyme responsible for the ovarian metabolism of progesterone to 20.alpha.-hydroxyprogesterone is 20.alpha.-hydroxysteroid dehydrogenase (20.alpha.-HSD). Specifically, 20.alpha.-HSD is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and catalyzes the transfer of hydrogen from NADPH to progesterone.
By metabolizing progesterone to an inactive form, 20.alpha.-HSD plays a central role in inhibiting the maintenance of pregnancy and prevention of implantation [Wiest, Endocrinology 83:1181-184 (1968); Wiest et al., Endocrinology 82:844-859 (1968); Kuhn and Briley, i Biochem. J. 0117:193-200 (1970); Rodway and Kuhn, Biochem. J. 152:433-443 (1975)]. Further supporting this role is the fact that it is the increase in ovarian 20.alpha.-HSD activity rather than a decrease in the synthesis of progesterone that contributes to the lower circulating progesterone levels associated with the termination of pregnancy [Kuhn and Briley, Biochem. J. 117:193-201 (1970)]. Indeed, 20.alpha.-HSD gene expression [Albarracin et al. Endocrinology 134:2453-2460 (1994)] and activity remains repressed throughout pregnancy but are induced before parturition [Wiest et al., Endocrinology 82:844-859 (1968); Kuhn and Briley, Biochem. J. 117:193-200 (1970]. Also, ovarian 20.alpha.-HSD catalyzes the decline in progesterone levels which occur during normal and induced termination of pregnancy and pseudopregnancy [Hashimoto and Wiest, Endocrinology 84:873-885 (1969); Naito et al., Endocrinology Jpn 33(1):43-50 (February 1986)].
While 20.alpha.-HSD is of much interest as a key enzyme in the termination/prevention of pregnancy, it is possible that the enzyme is also of importance in spontaneous abortions. Specifically, it is possible that a significant number of spontaneous abortions are due to early expression of 20.alpha.-HSD. Therefore, detection of early 20.alpha.-HSD expression would be of interest in those susceptible to early spontaneous abortions. If detection is made early enough, progesterone replacement therapy could be initiated to help maintain the pregnancy.
Since ovarian 20.alpha.-HSD has been identified as a key enzyme in the metabolism of progesterone, efforts have been undertaken to characterize the structural/functional aspects of the enzyme. However, these efforts have been hampered by the limited availability of the purified native form of the enzyme. Further, purified native enzyme is unsuitable for the studies designed to evaluate the role of post-translational events such as glycosylation and phosphorylation in enzyme activity. In order to avoid the limitations of using native 20.alpha.-HSD, cloning of the gene encoding the rat corpus luteum 20.alpha.-HSD has been undertaken and accomplished [Mao et al. Biochem. Biophys. Res. Comm. 201(3):1289-1295 (1994)]. The use of recombinant methods to produce 20.alpha.-HSD overcomes the limitations inherent in the use of native enzyme and provides an abundant source of the enzyme for a variety of uses.
These uses include, but are not limited to (1) obtaining a recombinant protein which can be used to inhibit the maintenance of pregnancy and/or prevent implantation leading to pregnancy and (2) obtain antibodies to the protein which can be used as a diagnostic agent to detect circulating levels of the protein in individuals at risk for spontaneous abortions.